Signaling mechanisms for stretch-dependent growth and differentiation of vascular smooth muscle were investigated in mechanically loaded rat portal veins in organ culture. Stretch-dependent protein synthesis was found to depend on endogenous release of angiotensin II. Autoradiography after [³⁵S]methionine incorporation revealed stretch-dependent synthesis of several proteins, of which SM22 and actin were particularly prominent. Inhibition of RhoA activity by cell-permeant C3 toxin increased tissue mechanical compliance and reduced stretch-dependent extracellular signal-regulated kinase (ERK)1/2 activation, growth, and synthesis of actin and SM22, suggesting a role of the actin cytoskeleton. In contrast, inhibition of Rho-associated kinase by Y-27632 did not reduce ERK1/2 phosphorylation or actin and SM22 synthesis and did not affect tissue mechanical compliance but still inhibited overall growth. The actin polymerization inhibitors latrunculin B and cytochalasin D both inhibited growth and caused increased tissue compliance. Whereas latrunculin B concentration-dependently reduced actin and SM22 synthesis, cytochalasin D did so at low (10⁻⁸ M) but not at high (10⁻⁶ M) concentration. The results show that stretch stabilizes the contractile smooth muscle phenotype. Stretch-dependent differentiation marker expression requires an intact cytoskeleton for stretch sensing, control of protein expression via the level of unpolymerized G-actin, or both.